Action Spectra

Photomed Laser Surg. 2008 Dec;26(6):593-9.

Absorption measurements of cell monolayers relevant to mechanisms of laser phototherapy: reduction or oxidation of cytochrome c oxidase under laser radiation at 632.8 nm.

Karu TI, Pyatibrat LV, Kolyakov SF, Afanasyeva NI.

Institute of Laser and Information Technologies of Russian Academy of Sciences, Troitsk, Moscow Region 142190, Russian Federation. tkaru@isan.troitsk.ru

Abstract

OBJECTIVE: The objective of this work was a further investigation of redox mechanisms of laser phototherapy on the cellular level.

BACKGROUND DATA: Cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, is believed to work as the photoacceptor to modulate cellular metabolism in laser phototherapy.

MATERIALS AND METHODS: The changes in the absorption spectra of HeLa-cell monolayers before and after irradiation at 632.8 nm using fast multi-channel recording were evaluated by the intensity ratio between the peaks at 770 and 670 nm (intensity ratio criterion).

RESULTS: By the intensity ratio criterion, the irradiation effects (reduction or oxidation of the photoacceptor) depended on the initial redox status of cytochrome c oxidase. The irradiation (three times at 632.8 nm, dose = 6.3 x 103 J/m(2), tau(irrad.) = 10 sec, tau(record.) = 600 msec) of cells initially characterized by relatively oxidized cytochrome c oxidase caused first a reduction of the photoacceptor, and then its oxidation (a bell-shaped curve). The irradiation by the same scheme of the cells with initially relatively reduced cytochrome c oxidase caused first oxidation and then a slight reduction of the enzyme (a curve opposite to the bell-shaped curve).

CONCLUSION: The experimental results of our work demonstrate that irradiation at 632.8 nm causes either a (transient) relative reduction of the photoacceptor, putatively cytochrome c oxidase, or its (transient) relative oxidation, depending on the initial redox status of the photoacceptor. The maximum in the bell-shaped dose-dependence curve or the minimum of the reverse curve is the turning point between the prevailing of oxidation or reduction processes. Our results are evidence that the bell-shaped dose dependences recorded for various cellular responses are characteristic also for redox changes in the photoacceptor, cytochrome c oxidase.

J Photochem Photobiol B. 2005 Nov 1;81(2):98-106. Epub 2005 Aug 26.

Absorption measurements of a cell monolayer relevant to phototherapy: reduction of cytochrome c oxidase under near IR radiation.

Karu TI, Pyatibrat LV, Kolyakov SF, Afanasyeva NI.

Institute of Laser and Information Technologies of Russian Academy of Sciences, Troitsk, Pionerskaya Street 2, Moscow Region 142190, Russian Federation. tkaru@isan.troitsk.ru

Phototherapy uses monochromatic light in the optical region of 600-1000 nm to treat in a non-destructive and non-thermal fashion various soft-tissue and neurological conditions. This kind of treatment is based on the ability of light red-to-near IR to alter cellular metabolism as a result of its being absorbed by cytochrome c oxidase. To further investigate the involvement of cytochrome c oxidase as a photoacceptor in the alteration of the cellular metabolism, we have aimed our study at, first, recording the absorption spectra of HeLa-cell monolayers in various oxygenation conditions (using fast multichannel recording), secondly, investigating the changes caused in these absorption spectra by radiation at 830 nm (the radiation wavelength often used in phototherapy), and thirdly, comparing between the absorption and action spectra recorded. The absorption measurements have revealed that the 710- to 790-nm spectral region is characteristic of a relatively reduced photoacceptor, while the 650- to 680-nm one characterizes a relatively oxidized photoacceptor. The ratio between the peak intensities at 760 and 665 nm is used to characterize the redox status of cytochrome c oxidase. By this criterion, the irradiation of the cellular monolayers with light at lambda=830 nm (D=6.3 x 10(3)J/m(2)) causes the reduction of the photoacceptor. A similarity is established between the peak positions at 616, 665, 760, 813, and 830 nm in the absorption spectra of the cellular monolayers and the action spectra of the long-term cellular responses (increase in the DNA synthesis rate and cell adhesion to a matrix).

Photomed Laser Surg. 2005 Aug;23(4):355-61.

Exact action spectra for cellular responses relevant to phototherapy.

Karu TI, Kolyakov SF.

Institute of Laser and Information Technologies, Russian Academy of Sciences, Troitsk, Moscow Region, Russian Federation. tkaru@isan.troitsk.ru

OBJECTIVE: The aim of the present work is to analyze available action spectra for various biological responses of HeLa cells irradiated with monochromatic light of 580-860 nm.

BACKGROUND DATA: Phototherapy (low-level laser therapy or photobiomodulation) is characterized by its ability to induce photobiological processes in cells. Exact action spectra are needed for determination of photoacceptors as well as for further investigations into cellular mechanisms of phototherapy. METHODS: Seven action spectra for the stimulation of DNA and RNA synthesis rate and cell adhesion to glass matrix are analyzed by curve fitting, followed by deconvolusion with Lorentzian fitting. Exact parameters of peak positions and bandwidths are presented.

RESULTS: The peak positions are between 613.5 and 623.5 nm (in one spectrum, at 606 nm), in the red maximum. The far-red maximum has exact peak positions between 667.5 and 683.7 nm in different spectra. Two near infrared maxima have peak positions in the range 750.7-772.3 nm and 812.5-846.0 nm, respectively.

CONCLUSIONS: In the wavelength range important for phototherapy (600-860 nm), there are four “active” regions, but peak positions are not exactly the same for all spectra.

Lasers Surg Med. 2005 Apr;36(4):307-14.

Cellular effects of low power laser therapy can be mediated by nitric oxide.

Karu TI, Pyatibrat LV, Afanasyeva NI.

Institute of Laser and Information Technologies of the Russian Academy of Sciences, 142190 Troitsk, Moscow, Russia. tkaru@isan.troitsk.ru

Abstract

BACKGROUND AND OBJECTIVES: The objective of this study was to investigate the possibility of involvement of nitric oxide (NO) into the irradiation-induced increase of cell attachment. These experiments were performed with a view to exploring the cellular mechanisms of low-power laser therapy.

STUDY DESIGN/MATERIALS AND METHODS: A suspension of HeLa cells was irradiated with a monochromatic visible-to-near infrared radiation (600-860 nm, 52 J/m2) or with a diode laser (820 nm, 8-120 J/m2) and the number of cells attached to a glass matrix was counted after 30 minute incubation at 37 degrees C. The NO donors sodium nitroprusside (SNP), glyceryl trinitrate (GTN), or sodium nitrite (NaNO2) in the concentration range 5 x 10(-9)-5 x 10(-4)M were added to the cellular suspension before or after irradiation. The action spectra and the concentration and fluence dependencies obtained were compared and analyzed.

RESULTS: The well-structured action spectrum for the increase of the adhesion of the cells, with maxima at 619, 657, 675, 740, 760, and 820 nm, points to the existence of a photoacceptor responsible for the enhancement of this property (supposedly cytochrome c oxidase, the terminal respiratory chain enzyme), as well as signaling pathways between the cell mitochondria, plasma membrane, and nucleus. Treating the cellular suspension with SNP (5 x 10(-5)M) before irradiation significantly modifies the action spectrum for the enhancement of the cell attachment property (band maxima at 642, 685, 700, 742, 842, and 856 nm). The action of SNP, GTN, and NaNO2 added before or after irradiation depends on their concentration and radiation fluence.

CONCLUSIONS: The NO donors added to the cellular suspension before irradiation eliminate the radiation-induced increase in the number of cells attached to the glass matrix, supposedly by way of binding NO to cytochrome c oxidase. NO added to the suspension after irradiation can also inhibit the light-induced signal downstream. Both effects of NO depend on the concentration of the NO donors added. These results indicate that NO can control the irradiation-activated reactions that increase the attachment of cells.

Int J Radiat Biol. 2000 Jun;76(6):863-70.

Specific helium-neon laser sensitivity of the purified cytochrome c oxidase.

Pastore D, Greco M, Passarella S.

Dipartimento di Scienze Animali, Vegetali e dell’Ambiente, Università del Molise, Campobasso, Italy.

Abstract

PURPOSE: In order to gain some insight into the mechanism of interaction between Helium-Neon (He-Ne) laser light and mitochondrial cytochromes, the sensitivity of cytochrome electron transfer activity to He-Ne laser was tested.

MATERIALS AND METHODS: Irradiation of solutions containing either purified cytochromes or dissolved rat liver mitochondria was carried out (wavelength 632.8 nm, fluence rate 10 mW cm(-2), fluence 2 J cm(-2)); the irradiation conditions were the ones able to affect cytochrome c oxidase (COX) activity in mitochondria (Pastore et al., 1994).

RESULTS: Cytochrome c oxidation catalysed by COX was affected by He-Ne laser irradiation of the purified enzyme. This result was obtained from measurements of the pseudo-first-order kinetic constant and from determinations of the turnover number of the enzyme, performed at different cytochrome c/COX ratios. Consistently, the kinetic parameters of COX changed. On the contrary, no alteration in the rate of electron transfer catalysed by either cytochrome c or bc1 complex was found.

CONCLUSIONS: This study shows that purified COX is a specific target of He-Ne laser light; therefore, COX may be considered to be a mitochondrial photo-acceptor.

J Photochem Photobiol B. 1999 Mar;49(1):1-17.

Primary and secondary mechanisms of action of visible to near-IR radiation on cells.

Karu T.

Laser Technology Research Center of Russian Academy of Sciences, Troitsk, Moscow Region, Russia. kara@isan.troitsk.ru

Cytochrome c oxidase is discussed as a possible photoacceptor when cells are irradiated with monochromatic red to near-IR radiation. Four primary action mechanisms are reviewed: changes in the redox properties of the respiratory chain components following photoexcitation of their electronic states, generation of singlet oxygen, localized transient heating of absorbing chromophores, and increased superoxide anion production with subsequent increase in concentration of the product of its dismutation, H2O2. A cascade of reactions connected with alteration in cellular homeostasis parameters (pHi, [Cai], cAMP, Eh, [ATP] and some others) is considered as a photosignal transduction and amplification chain in a cell (secondary mechanisms).